Comparative Study of Latanoprost Drug Delivery Systems for Glaucoma Treatment and Their Interaction with the Tear Film Lipid Layer Models.

This study investigates the interaction of two approved and one newly developed latanoprost formulation with in vitro and in silico models of the tear film and tear film lipid layer (TFLL). Latanoprost, a prostaglandin analogue used for intraocular elevated pressure treatment, is topically delivered by nanocarriers within aqueous solutions or emulsions. The study focuses on the impact of these carriers on drug interactions with the tear film and their effect on the TFLL. Three different types of latanoprost carriers, micellar, nanoemulsion, and polymer-based, were compared, and each revealed distinct interaction patterns with the TFLL. Surface pressure kinetics demonstrated a rapid increase for the benzalkonium chloride formulation and a slow rise for the preservative-free variants. Visualization of the acellular in vitro TFLL model revealed different patterns of incorporation for each formulation, indicating unique interaction mechanisms. Molecular dynamics simulations further revealed different mechanisms of drug release in the TFLL between micellar and nanoemulsion formulations. In-depth examination highlighted the role of triglyceride molecules in replenishing the nonpolar layer of the TFLL, which suggests potential improvements in ocular surface compatibility by adjusting the quality and concentration of the oily phase. These findings suggest the potential for optimizing latanoprost formulations by tuning the oily phase-to-surfactant ratio and selecting suitable surfactants.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

The Potential Role of SP-G as Surface Tension Regulator in Tear Film: From Molecular Simulations to Experimental Observations.

The ocular surface is in constant interaction with the environment and with numerous pathogens. Therefore, complex mechanisms such as a stable tear film and local immune defense mechanisms are required to protect the eye. This study describes the detection, characterization, and putative role of surfactant protein G (SP-G/SFTA2) with respect to wound healing and surface activity. Bioinformatic, biochemical, and immunological methods were combined to elucidate the role of SP-G in tear film. The results show the presence of SP-G in ocular surface tissues and tear film (TF). Increased expression of SP-G was demonstrated in TF of patients with dry eye disease (DED). Addition of recombinant SP-G in combination with lipids led to an accelerated wound healing of human corneal cells as well as to a reduction of TF surface tension. Molecular modeling of TF suggest that SP-G may regulate tear film surface tension and improve its stability through specific interactions with lipids components of the tear film. In conclusion, SP-G is an ocular surface protein with putative wound healing properties that can also reduce the surface tension of the tear film.

Structural Effects of Cation Binding to DPPC Monolayers.

Ions at the two sides of the plasma membrane maintain the transmembrane potential, participate in signaling, and affect the properties of the membrane itself. The extracellular leaflet is particularly enriched in phosphatidylcholine lipids and under the influence of Na+, Ca2+, and Cl- ions. In this work, we combined molecular dynamics simulations performed using state-of-the-art models with vibrational sum frequency generation (VSFG) spectroscopy to study the effects of these key ions on the structure of dipalmitoylphosphatidylcholine. We used lipid monolayers as a proxy for membranes, as this approach enabled a direct comparison between simulation and experiment. We find that the effects of Na+ are minor. Ca2+, on the other hand, strongly affects the lipid headgroup conformations and induces a tighter packing of lipids, thus promoting the liquid condensed phase. It does so by binding to both the phosphate and carbonyl oxygens via direct and water-mediated binding modes, the ratios of which depend on the monolayer packing. Clustering analysis performed on simulation data revealed that changes in area per lipid or CaCl2 concentration both affect the headgroup conformations, yet their effects are anticorrelated. Cations at the monolayer surface also attract Cl-, which at large CaCl2 concentrations penetrates deep to the monolayer. This phenomenon coincides with a radical change in the VSFG spectra of the phosphate group, thus indicating the emergence of a new binding mode.

Properties of Lipid Models of Lung Surfactant Containing Cholesterol and Oxidized Lipids: A Mixed Experimental and Computational Study.

We introduce and study a multicomponent lipid film mimicking lipid composition of the human lung surfactant. It consists of phospholipids with various lipid headgroups and tail saturation. Furthermore, it includes cholesterol and oxidized lipids. Langmuir trough and fluorescence microscopy experiments are combined with fully atomistic molecular dynamics simulations. The considered lipid mixtures form complex interfacial films with properties modulated by lateral compression. Cholesterol laterally condenses, and oxidized lipids laterally expand the films; both types of molecules increase film miscibility. Oxidized lipids also alter the lipid-water interface enhancing film hydration; this effect can be partially reversed by cholesterol. Regarding presentation of different chemical moieties toward the aqueous subphase, the zwitterionic phosphatidylcholine groups dominate at the lipid-water interface, while both the negatively charged phosphatidylglycerol and hydroxyl group of cholesterol are less exposed. The investigated synthetic lipid-only mimic of the lung surfactant may serve as a basis for further studies involving nonlipid pulmonary surfactant components.

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Membrane Lipid Nanodomains.

Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.

Improving Stability of Tear Film Lipid Layer via Concerted Action of Two Drug Molecules: A Biophysical View.

The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.

Concurrent Compression of Phospholipid Membranes by Calcium and Cholesterol.

Regulation of cell metabolism, membrane fusion, association of proteins with cellular membranes, and cellular signaling altogether would not be possible without Ca2+ ions. The distribution of calcium within the cell is uneven with the negatively charged inner leaflet of the plasma membrane being one of the primary targets of its accumulation. Therefore, we decided to map the influence of Ca2+ on the properties of lipid bilayers closely resembling natural lipid membranes. We combined fluorescence spectroscopy (analysis of time-resolved emission spectra of Laurdan probe and derived parameters: integrated relaxation time related to local lipid mobility, and total emission shift reflecting membrane polarity and hydration) with molecular dynamics simulations to determine the effect of the increasing CaCl2 concentration on model lipid membranes containing POPC, POPS, and cholesterol. On top of that, the impact of calcium on the plasma membranes isolated from HEK293 cells was investigated using the steady-state fluorescence of Laurdan. We found that calcium increases rigidity of all the model lipid membranes used, elevates their thickness, increases lipid packing and ordering, and impedes the local lipid mobility. All these effects were to a great extent similar to those elicited by cholesterol. However, the changes of the membrane properties induced by calcium and cholesterol seem largely independent from each other. At sufficiently high concentrations of calcium or cholesterol, the steric effects hindered a further alteration of membrane organization, i.e., the compressibility limit of membrane structures was reached. We found no indication for mutual interaction between Ca2+ and cholesterol, nor competition of Ca2+ ions and hydroxyl groups of cholesterol for binding to phospholipids. Fluorescence measurements indicated that Ca2+ adsorption decreases mobility within the carbonyl region of model bilayers more efficiently than monovalent ions do (Ca2+ ≫ Li+ > Na+ > K+ > Cs+). The effects of calcium ions were to a great extent mitigated in the plasma membranes isolated from HEK293 cells when compared to the model lipid membranes. Noticeably, the plasma membranes showed remarkably higher resistance toward rigidification induced by calcium ions even when compared with the model membranes containing cholesterol.

Atomistic Model for Nearly Quantitative Simulations of Langmuir Monolayers.

Lung surfactant and a tear film lipid layer are examples of biologically relevant macromolecular structures found at the air-water interface. Because of their complexity, they are often studied in terms of simplified lipid layers, the simplest example being a Langmuir monolayer. Given the profound biological significance of these lipid assemblies, there is a need to understand their structure and dynamics on the nanoscale, yet there are not many techniques able to provide this information. Atomistic molecular dynamics simulations would be a tool fit for this purpose; however, the simulation models suggested until now have been qualitative instead of quantitative. This limitation has mainly stemmed from the challenge to correctly describe the surface tension of water with simulation parameters compatible with other biomolecules. In this work, we show that this limitation can be overcome by using the recently introduced four-point OPC water model, whose surface tension for water is demonstrated to be quantitatively consistent with experimental data and which is also shown to be compatible with the commonly employed lipid models. We further establish that the approach of combining the OPC four-point water model with the CHARMM36 lipid force field provides nearly quantitative agreement with experiments for the surface pressure-area isotherm for POPC and DPPC monolayers, also including the experimentally observed phase coexistence in a DPPC monolayer. The simulation models reported in this work pave the way for nearly quantitative atomistic studies of lipid-rich biological structures at air-water interfaces.

Orientation of Laurdan in Phospholipid Bilayers Influences Its Fluorescence: Quantum Mechanics and Classical Molecular Dynamics Study.

Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.

Tear film lipid layer: A molecular level view.

Human cornea is covered by an aqueous tear film, and the outermost layer of the tear film is coated by lipids. This so-called tear film lipid layer (TFLL) reduces surface tension of the tear film and helps with the film re-spreading after blinks. Alterations of tear lipids composition and properties are related to dry eye syndrome. Therefore, unveiling structural and functional properties of TFLL is necessary for understanding tear film function under both normal and pathological conditions. Key properties of TFLL, such as resistance against high lateral pressures and ability to spread at the tear film surface, are directly related to the chemical identity of TFLL lipids. Hence, a molecular-level description is required to get better insight into TFLL properties. Molecular dynamics simulations are particularly well suited for this task and they were recently used for investigating TFLL. The present review discusses molecular level organization and properties of TFLL as seen by these simulation studies. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Mixed DPPC/POPC Monolayers: All-atom Molecular Dynamics Simulations and Langmuir Monolayer Experiments.

To elucidate the consequences of the saturated-unsaturated nature of lipid surface films, monolayers formed by an equimolar mixture of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids are investigated in a wide range of surface pressures. As such mixtures share some features with naturally-occurring surfactants, for example the lung surfactant, the systems are studied at the temperature relevant for human body. All-atom molecular dynamics simulations and Langmuir trough experiments are employed. The binary lipid mixture is compared with the corresponding one-component systems. Atomistic-level alterations of monolayer molecular properties upon lateral compression are scrutinized. These involve elevation of lateral ordering of lipid chains, modulation of chain and headgroup orientation, and reduction of lipid hydration. The presence of the unsaturated POPC in the DPPC/POPC mixture reduces the liquid expanded-liquid condensed coexistence region and moderates the phase transition. Simulations predict that nanoscale lipid de-mixing occurs with small transient DPPC clusters emerging due to local fluctuations of the lateral lipid arrangement. A vertical sorting of lipids induced by lateral compression is also observed, with DPPC transferred toward the water phase. Both the conformational lipid alterations due to monolayer compression as well as the existence of lateral dynamic inhomogeneities of the lipid film are potentially pertain to dynamic and non-homogeneous lipid interfacial systems.

Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition.

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P2 containing liposomes reveal that Ca2+ as well as Mg2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study.

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates.

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

Modeling Lung Surfactant Interactions with Benzo[a]pyrene.

By reducing the surface tension of the air-water interface in alveoli, lung surfactant (LS) is crucial for proper functioning of the lungs. It also forms the first barrier against inhaled pathogens. In this study we inspect the interactions of LS models with a dangerous air pollutant, benzo[a]pyrene (BaP). Dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoylphosphatidylcholine, and their 1:1 mixture are used as LS models. Pressure-area isotherms are employed to study macroscopic properties of the monolayers. We find that addition of BaP has a condensing effect, manifested by lowering the values of surface pressure and shifting the isotherms to smaller areas. Atomistic details of this process are examined by means of molecular dynamics simulations. We show that initially BaP molecules are accumulated in the monolayers. Upon compression, they are forced to the headgroups region and eventually expelled to the subphase. BaP presence results in reduction of monolayer hydration in the hydrophilic region. In the hydrophobic region it induces increased chain ordering, reduction of monolayer fluidity, and advances transition to the liquid condensed phase in the DPPC system.

Exploring Fluorescent Dyes at Biomimetic Interfaces with Second Harmonic Generation and Molecular Dynamics.

The adsorption of a DNA fluorescent probe belonging to the thiazole orange family at the dodecane/water and dodecane/phospholipid/water interfaces has been investigated using a combination of surface second harmonic generation (SSHG) and all-atomistic molecular dynamics (MD) simulations. Both approaches point to a high affinity of the cationic dye for the dodecane/water interface with a Gibbs free energy of adsorption on the order of -45 kJ/mol. Similar affinity was observed with a monolayer of negatively charged DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)) lipids. On the other hand, no significant adsorption could be found with the zwitterionic DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) lipids. This was rationalized in terms of Coulombic interactions between the monolayer surface and the cationic dye. The similar affinity for the interface with and without DPPG, despite the favorable Coulombic attraction in the latter case, could be explained after investigating the interfacial orientation of the dye. In the absence of a monolayer, the dye adsorbs with its molecular plane almost flat at the interface, whereas in the presence of DPPG it has to intercalate into the monolayer and adopt a significantly different orientation to benefit from the electrostatic stabilization.

Membrane targeting of the yeast exocyst complex.

The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.

Comprehensive portrait of cholesterol containing oxidized membrane.

Biological membranes are under significant oxidative stress caused by reactive oxygen species mostly originating during cellular respiration. Double bonds of the unsaturated lipids are most prone to oxidation, which might lead to shortening of the oxidized chain and inserting of terminal either aldehyde or carboxylic group. Structural rearrangement of oxidized lipids, addressed already, is mainly associated with looping back of the hydrophilic terminal group. This contribution utilizing dual-focus fluorescence correlation spectroscopy and electron paramagnetic resonance as well as atomistic molecular dynamics simulations focuses on the overall changes of the membrane structural and dynamical properties once it becomes oxidized. Particularly, attention is paid to cholesterol rearrangement in the oxidized membrane revealing its preferable interaction with carbonyls of the oxidized chains. In this view cholesterol seems to have a tendency to repair, rather than condense, the bilayer.